A high-resolution map of functional miR-181 response elements in the thymus reveals the role of coding sequence targeting and an alternative seed match.

MicroRNAs (miRNAs) are critical post-transcriptional regulators in many biological processes. They act by guiding RNA-induced silencing complexes to miRNA response elements (MREs) in target mRNAs, inducing translational inhibition and/or mRNA degradation. Functional MREs are expected to predominantly occur in the 3' untranslated region and involve perfect base-pairing of the miRNA seed. Here, we generate a high-resolution map of miR-181a/b-1 (miR-181) MREs to define the targeting rules of miR-181 in developing murine T cells. By combining a multi-omics approach with computational high-resolution analyses, we uncover novel miR-181 targets and demonstrate that miR-181 acts predominantly through RNA destabilization. Importantly, we discover an alternative seed match and identify a distinct set of targets with repeat elements in the coding sequence which are targeted by miR-181 and mediate translational inhibition. In conclusion, deep profiling of MREs in primary cells is critical to expand physiologically relevant targetomes and establish context-dependent miRNA targeting rules.

Liver-specific deletion of miR-181ab1 reduces liver tumour progression via upregulation of CBX7.

MiR-181 expression levels increased in hepatocellular carcinoma (HCC) compared to non-cancerous tissues. MiR-181 has been widely reported as a possible driver of tumourigenesis but also acts as a tumour suppressor. In addition, the miR-181 family regulates the development and function of immune and vascular cells, which play vital roles in the progression of tumours. More complicatedly, many genes have been identified as miR-181 targets to mediate the effects of miR-181. However, the role of miR-181 in the development of primary tumours remains largely unexplored. We aimed to examine the function of miR-181 and its vital mediators in the progression of diethylnitrosamine-induced primary liver cancers in mice. The size of liver tumours was significantly reduced by 90% in global (GKO) or liver-specific (LKO) 181ab1 knockout mice but not in hematopoietic and endothelial lineage-specific knockout mice, compared to WT mice. In addition, the number of tumours was significantly reduced by 50% in GKO mice. Whole-genome RNA-seq analysis and immunohistochemistry showed that epithelial-mesenchymal transition was partially reversed in GKO tumours compared to WT tumours. The expression of CBX7, a confirmed miR-181 target, was up-regulated in GKO compared to WT tumours. Stable CBX7 expression was achieved with an AAV/Transposase Hybrid-Vector System and up-regulated CBX7 expression inhibited liver tumour progression in WT mice. Hepatic CBX7 deletion restored the progression of LKO liver tumours. MiR-181a expression was the lowest and CBX7 expression the highest in iClust2 and 3 subclasses of human HCC compared to iClust1. Gene expression profiles of GKO tumours overlapped with low-proliferative peri-portal-type HCCs. Liver-specific loss of miR-181ab1 inhibited primary liver tumour progression via up-regulating CBX7 expression, but tumour induction requires both hepatic and non-hepatic miR-181. Also, miR-181ab1-deficient liver tumours may resemble low-proliferative periportal-type human HCC. miR-181 was increased with liver tumour growth. More miR-181, darker colour and higher shape. CBX7 was very low in pericentral hepatocytes, increased in early liver tumours, but reduced in advanced liver tumours. Its levels were maintained in miR-181 KO liver tumours. In tumours (T), brown (darker is more) represents miR-181, the blue circle (thicker is more) represents CBX7.

miR-181a/b-1 controls thymic selection of Treg cells and tunes their suppressive capacity.

The interdependence of selective cues during development of regulatory T cells (Treg cells) in the thymus and their suppressive function remains incompletely understood. Here, we analyzed this interdependence by taking advantage of highly dynamic changes in expression of microRNA 181 family members miR-181a-1 and miR-181b-1 (miR-181a/b-1) during late T-cell development with very high levels of expression during thymocyte selection, followed by massive down-regulation in the periphery. Loss of miR-181a/b-1 resulted in inefficient de novo generation of Treg cells in the thymus but simultaneously permitted homeostatic expansion in the periphery in the absence of competition. Modulation of T-cell receptor (TCR) signal strength in vivo indicated that miR-181a/b-1 controlled Treg-cell formation via establishing adequate signaling thresholds. Unexpectedly, miR-181a/b-1-deficient Treg cells displayed elevated suppressive capacity in vivo, in line with elevated levels of cytotoxic T-lymphocyte-associated 4 (CTLA-4) protein, but not mRNA, in thymic and peripheral Treg cells. Therefore, we propose that intrathymic miR-181a/b-1 controls development of Treg cells and imposes a developmental legacy on their peripheral function.

Development of interleukin-17-producing γδ T cells is restricted to a functional embryonic wave.

γδ T cells are an important innate source of interleukin-17 (IL-17). In contrast to T helper 17 (Th17) cell differentiation, which occurs in the periphery, IL-17-producing γδ T cells (γδT17 cells) are probably committed during thymic development. To study when γδT17 cells arise during ontogeny, we used TcrdH2BeGFP reporter mice to monitor T cell receptor (TCR) rearrangement and IL-17 production in the embryonic thymus. We observed that several populations such as innate lymphoid cells and early T cell precursors were able to produce IL-17 prior to (and thus independent of) TCR recombination. γδT17 cells were absent after transplantation of IL-17-sufficient bone marrow into mice lacking both Il17a and Il17f. Also, γδT17 cells were not generated after genetic restoration of defective Rag1 function in adult mice. Together, these data suggested that these cells developed exclusively before birth and subsequently persisted in adult mice as self-renewing, long-lived cells.

Modeling the Dynamics of T-Cell Development in the Thymus.

The thymus hosts the development of a specific type of adaptive immune cells called T cells. T cells orchestrate the adaptive immune response through recognition of antigen by the highly variable T-cell receptor (TCR). T-cell development is a tightly coordinated process comprising lineage commitment, somatic recombination of Tcr gene loci and selection for functional, but non-self-reactive TCRs, all interspersed with massive proliferation and cell death. Thus, the thymus produces a pool of T cells throughout life capable of responding to virtually any exogenous attack while preserving the body through self-tolerance. The thymus has been of considerable interest to both immunologists and theoretical biologists due to its multi-scale quantitative properties, bridging molecular binding, population dynamics and polyclonal repertoire specificity. Here, we review experimental strategies aimed at revealing quantitative and dynamic properties of T-cell development and how they have been implemented in mathematical modeling strategies that were reported to help understand the flexible dynamics of the highly dividing and dying thymic cell populations. Furthermore, we summarize the current challenges to estimating in vivo cellular dynamics and to reaching a next-generation multi-scale picture of T-cell development.

miR-191 modulates B-cell development and targets transcription factors E2A, Foxp1, and Egr1.

The interdependence of posttranscriptional gene regulation via miRNA and transcriptional regulatory networks in lymphocyte development is poorly understood. Here, we identified miR-191 as direct upstream modulator of a transcriptional module comprising the transcription factors Foxp1, E2A, and Egr1. Deletion as well as ectopic expression of miR-191 resulted in developmental arrest in B lineage cells, indicating that fine tuning of the combined expression levels of Foxp1, E2A, and Egr1, which in turn control somatic recombination and cytokine-driven expansion, constitutes a prerequisite for efficient B-cell development. In conclusion, we propose that miR-191 acts as a rheostat in B-cell development by fine tuning a key transcriptional program.

Vascular miR-181b controls tissue factor-dependent thrombogenicity and inflammation in type 2 diabetes.

BACKGROUND: Diabetes mellitus is characterized by chronic vascular inflammation leading to pathological expression of the thrombogenic full length (fl) tissue factor (TF) and its isoform alternatively-spliced (as) TF. Blood-borne TF promotes factor (F) Xa generation resulting in a pro-thrombotic state and cardiovascular complications. MicroRNA (miR)s impact gene expression on the post-transcriptional level and contribute to vascular homeostasis. Their distinct role in the control of the diabetes-related procoagulant state remains poorly understood. METHODS: In a cohort of patients with poorly controlled type 2 diabetes (n = 46) plasma levels of miR-181b were correlated with TF pathway activity and markers for vascular inflammation. In vitro, human microvascular endothelial cells (HMEC)-1 and human monocytes (THP-1) were transfected with miR-181b or anti-miR-181b and exposed to tumor necrosis factor (TNF) α or lipopolysaccharides (LPS). Expression of TF isoforms, vascular adhesion molecule (VCAM) 1 and nuclear factor (NF) κB nuclear translocation was assessed. Moreover, aortas, spleen, plasma, and bone marrow-derived macrophage (BMDM)s of mice carrying a deletion of the first miR-181b locus were analyzed with respect to TF expression and activity. RESULTS: In patients with type 2 diabetes, plasma miR-181b negatively correlated with the procoagulant state as evidenced by TF protein, TF activity, D-dimer levels as well as markers for vascular inflammation. In HMEC-1, miR-181b abrogated TNFα-induced expression of flTF, asTF, and VCAM1. These results were validated using the anti-miR-181b. Mechanistically, we confirmed a miR-181b-mediated inhibition of importin-α3 (KPNA4) leading to reduced nuclear translocation of the TF transcription factor NFκB. In THP-1, miR-181b reduced both TF isoforms and FXa generation in response to LPS due to targeting phosphatase and tensin homolog (PTEN), a principal inducer for TF in monocytes. Moreover, in miR-181-/- animals, we found that reduced levels of miR-181b were accompanied by increased TF, VCAM1, and KPNA4 expression in aortic tissue as well as increased TF and PTEN expression in spleen. Finally, BMDMs of miR-181-/- mice showed increased TF expression and FXa generation upon stimulation with LPS. CONCLUSIONS: miR-181b epigenetically controls the procoagulant state in diabetes. Reduced miR-181b levels contribute to increased thrombogenicity and may help to identify individuals at particular risk for thrombosis.

T Cell Development by the Numbers.

T cells are continually generated in the thymus in a highly dynamic process comprising discrete steps of lineage commitment, T cell receptor (TCR) gene rearrangement, and selection. These steps are linked to distinct rates of proliferation, survival, and cell death, but a quantitative picture of T cell development is only beginning to emerge. Here we summarize recent technical advances, including genetic fate mapping, barcoding, and molecular timers, that have allowed the implementation of computational models to quantify developmental dynamics in the thymus. Coupling new techniques with mathematical models has recently resulted in the emergence of new paradigms in early hematopoiesis and might similarly open new perspectives on T cell development.

MicroRNA miR-181-A Rheostat for TCR Signaling in Thymic Selection and Peripheral T-Cell Function.

The selection of T cells during intra-thymic d evelopment is crucial to obtain a functional and simultaneously not self-reactive peripheral T cell repertoire. However, selection is a complex process dependent on T cell receptor (TCR) thresholds that remain incompletely understood. In peripheral T cells, activation, clonal expansion, and contraction of the active T cell pool, as well as other processes depend on TCR signal strength. Members of the microRNA (miRNA) miR-181 family have been shown to be dynamically regulated during T cell development as well as dependent on the activation stage of T cells. Indeed, it has been shown that expression of miR-181a leads to the downregulation of multiple phosphatases, implicating miR-181a as ''rheostat'' of TCR signaling. Consistently, genetic models have revealed an essential role of miR-181a/b-1 for the generation of unconventional T cells as well as a function in tuning TCR sensitivity in peripheral T cells during aging. Here, we review these broad roles of miR-181 family members in T cell function via modulating TCR signal strength.

MicroRNA miR-181a/b-1 controls MAIT cell development.

Mucosal-associated invariant T (MAIT) cells constitute a major fraction of innate-like T cells in humans with critical roles in defense against microbial pathogens and in maintaining mucosal integrity. However, the molecular mechanisms underlying MAIT cell development remain largely elusive. Here we investigated the role of miR-181a/b-1, a pair of microRNAs that serve as rheostat of TCR signal strength, in this process. Loss of miR-181a/b-1 in mice resulted in a profound arrest in early MAIT cell development. As a consequence, in the absence of miR-181a/b-1, thymic MAIT cells failed to acquire functional maturity based on expression of transcription factors PLZF, T-bet and RORγt. Temporal analysis of development using a molecular timer in combination with loss of miR-181a/b-1 revealed that MAIT cells complete functional maturation in the periphery and indicates that functionally mature MAIT cells in the thymus are long-term resident cells. Thus, our study provides insight into the dynamics of MAIT cell development in vivo. Of note, deletion of miR-181a/b-1 alone completely mirrored loss of all miRNAs.

miR-181a Expression in Donor T Cells Modulates Graft-versus-Host Disease after Allogeneic Bone Marrow Transplantation.

Because miR-181a has been described to alter T cell activation, we hypothesized that manipulation of miR-181a expression in donor T cells may alter acute graft-versus-host disease (aGvHD) after allogeneic bone marrow transplantation (BMT). We therefore analyzed the impact of enhanced and reduced miR-181a expression in donor T cells on aGvHD induction by lentiviral gene transfer into primary T cells and using miR-181a/b-1(-/-) T cells, respectively. BMT-recipient mice receiving donor T cells with enhanced miR-181a expression showed no signs of aGvHD and survived for the time of follow-up, whereas T cells lacking miR-181a/b-1 accelerated aGvHD. In line with these data, analysis of donor T cells in blood, secondary lymphoid organs, and target organs of aGvHD after BMT showed significantly reduced numbers of miR-181a-transduced T cells, as compared with controls. In addition, expansion of activated T cells with enhanced miR-181a expression was reduced in vitro and in vivo. We further show that anti-apoptotic BCL-2 protein expression is reduced in murine and human T cells upon overexpression of miR-181a, suggesting that regulation of BCL-2-expression by miR-181a may contribute to altered alloreactivity of T cells in aGvHD. These data indicate that proteins regulated by miR-181a may be therapeutic targets for aGvHD prevention.

Limited niche availability suppresses murine intrathymic dendritic-cell development from noncommitted progenitors.

The origins of dendritic cells (DCs) and other myeloid cells in the thymus have remained controversial. In this study, we assessed developmental relationships between thymic dendritic cells and thymocytes, employing retrovirus-based cellular barcoding and reporter mice, as well as intrathymic transfers coupled with DC depletion. We demonstrated that a subset of early T-lineage progenitors expressed CX3CR1, a bona fide marker for DC progenitors. However, intrathymic transfers into nonmanipulated mice, as well as retroviral barcoding, indicated that thymic dendritic cells and thymocytes were largely of distinct developmental origin. In contrast, intrathymic transfers after in vivo depletion of DCs resulted in intrathymic development of non-T-lineage cells. In conclusion, our data support a model in which the adoption of T-lineage fate by noncommitted progenitors at steady state is enforced by signals from the thymic microenvironment unless niches promoting alternative lineage fates become available.

Responsiveness of Developing T Cells to IL-7 Signals Is Sustained by miR-17∼92.

miRNAs regulate a large variety of developmental processes including development of the immune system. T cell development is tightly controlled through the interplay of transcriptional programs and cytokine-mediated signals. However, the role of individual miRNAs in this process remains largely elusive. In this study, we demonstrated that hematopoietic cell-specific loss of miR-17∼92, a cluster of six miRNAs implicated in B and T lineage leukemogenesis, resulted in profound defects in T cell development both at the level of prethymic T cell progenitors as well as intrathymically. We identified reduced surface expression of IL-7R and concomitant limited responsiveness to IL-7 signals as a common mechanism resulting in reduced cell survival of common lymphoid progenitors and thymocytes at the double-negative to double-positive transition. In conclusion, we identified miR-17∼92 as a critical modulator of multiple stages of T cell development.

Overexpression of Vα14Jα18 TCR promotes development of iNKT cells in the absence of miR-181a/b-1.

Expression of microRNA miR-181a/b-1 is critical for intrathymic development of invariant natural killer T (iNKT) cells. However, the underlying mechanism has remained a matter of debate. On the one hand, growing evidence suggested that miR-181a/b-1 is instrumental in setting T-cell receptor (TCR) signaling threshold and thus permits agonist selection of iNKT cells through high-affinity TCR ligands. On the other hand, alterations in metabolic fitness mediated by miR-181a/b-1-dependent dysregulation of phosphatase and tensin homolog (Pten) have been proposed to cause the iNKT-cell defect in miR-181-a/b-1-deficient mice. To re-assess the hypothesis that modulation of TCR signal strength is the key mechanism by which miR-181a/b-1 controls the development of iNKT cells, we generated miR-181a/b-1-deficient mice expressing elevated levels of a Vα14Jα18 TCRα chain. In these mice, development of iNKT cells was fully restored. Furthermore, both subset distribution of iNKT cells as well as TCR Vß repertoire were independent of the presence of miR-181a/b-1 once a Vα14Jα18 TCRα chain was overexpressed. Finally, levels of Pten protein were similar in Vα14Jα18 transgenic mice irrespective of their miR-181a/b-1 status. Collectively, our data support a model in which miR-181 promotes development of iNKT cells primarily by generating a permissive state for agonist selection with alterations in metabolic fitness possibly constituting a secondary effect.

Immunoglobulins drive terminal maturation of splenic dendritic cells.

Nature and physiological status of antigen-presenting cells, such as dendritic cells DCs, are decisive for the immune reactions elicited. Multiple factors and cell interactions have been described that affect maturation of DCs. Here, we show that DCs arising in the absence of immunoglobulins (Ig) in vivo are impaired in cross-presentation of soluble antigen. This deficiency was due to aberrant cellular targeting of antigen to lysosomes and its rapid degradation. Function of DCs could be restored by transfer of Ig irrespective of antigen specificity and isotype. Modulation of cross-presentation by Ig was inhibited by coapplication of mannan and, thus, likely to be mediated by C-type lectin receptors. This unexpected dependency of splenic DCs on Ig to cross-present antigen provides insights into the interplay between cellular and humoral immunity and the immunomodulatory capacity of Ig.

Critical role for miR-181a/b-1 in agonist selection of invariant natural killer T cells.

T-cell receptor (TCR) signal strength determines selection and lineage fate at the CD4(+)CD8(+) double-positive stage of intrathymic T-cell development. Members of the miR-181 family constitute the most abundantly expressed microRNA at this stage of T-cell development. Here we show that deletion of miR-181a/b-1 reduced the responsiveness of double-positive thymocytes to TCR signals and virtually abrogated early invariant natural killer T (iNKT) cell development, resulting in a dramatic reduction in iNKT cell numbers in thymus as well as in the periphery. Increased concentrations of agonist ligand rescued iNKT cell development in miR-181a/b-1(-/-) mice. Our results define a critical role of miR-181a/b-1 in early iNKT cell development and show that miR-181a/b-1 sets a TCR signaling threshold for agonist selection.

B-cell modulation of dendritic-cell function: signals from the far side.

An appropriate immune response against a specific pathogen requires finely orchestrated interactions between the various cell populations within the immune system. At the same time, immunological tolerance to self must be maintained. DCs play an essential role in achieving these dual requisites. They coordinate adaptive immunity by integrating signals directly emanating from both infectious agents and cells of the immune system. Many such signals, especially those from innate cells and T cells, have been extensively characterized. In contrast, little is known about how B cells modulate function of DCs. B cells produce a variety of cytokines, including IL-10 and IL-6, which are known to influence DC function. In addition, Igs constitute the major secretory products of terminally differentiated B cells (plasma cells). DCs express various types of receptors for binding Ig, such as Fc receptors and C-type lectin receptors. In accordance, Igs can regulate DC function depending on the receptors engaged. Here, we review the emerging immunomodulatory role of cytokines and Ig secreted by B cells. We discuss the evidence for how these B-cell-derived factors may shape the adaptive immune response by directly acting on DCs.

Human dendritic cell interactions with the zoonotic parasite Cryptosporidium parvum result in activation and maturation.

Cryptosporidiosis in humans is caused by infection of the zoonotic apicomplexan parasite Cryptosporidium parvum. In 2006, it was included by the World Health Organization (WHO) in the group of the most neglected poverty-related diseases. It is characterized by enteritis accompanied by profuse catarrhalic diarrhea with high morbidity and mortality, especially in children of developing countries under the age of 5 years and in HIV patients. The vulnerability of HIV patients indicates that a robust adaptive immune response is required to successfully fight this parasite. Little is known, however, about the adaptive immune response against C. parvum. To have an insight into the early events of the adaptive immune response, we generated primary human dendritic cells (DCs) from monocytes of healthy blood donors and exposed them to C. parvum oocysts and sporozoites in vitro. DCs are equipped with numerous receptors that detect microbial molecules and alarm signals. If stimulation is strong enough, an essential maturation process turns DCs into unique activators of naïve T cells, a prerequisite of any adaptive immune response. Parasite exposure highly induced the production of the pro-inflammatory cytokines/chemokines interleukin (IL)-6 and IL-8 in DCs. Moreover, antigen-presenting molecules (HLA-DR and CD1a), maturation markers, and costimulatory molecules required for T-cell stimulation (CD83, CD40, and CD86) and adhesion molecules (CD11b and CD58) were all upregulated. In addition, parasite-exposed human DCs showed enhanced cell adherence, increased mobility, and a boosted but time-limited phagocytosis of C. parvum oocysts and sporozoites, representing other prerequisites for antigen presentation. Unlike several other microbial stimuli, C. parvum exposure rather led to increased oxidative consumption rates (OCRs) than extracellular acidification rates (ECARs) in DCs, indicating that different metabolic pathways were used to provide energy for DC activation. Taken together, C. parvum-exposed human DCs showed all hallmarks of successful maturation, enabling them to mount an effective adaptive immune response.

Cytotaxonomic characterization and estimation of migration patterns of onchocerciasis vectors (Simulium damnosum sensu lato) in northwestern Ethiopia based on RADSeq data.

BACKGROUND: While much progress has been made in the control and elimination of onchocerciasis across Africa, the extent to which vector migration might confound progress towards elimination or result in re-establishment of endemism in areas where transmission has been eliminated remains unclear. In Northern Ethiopia, Metema and Metekel-two foci located near the Sudan border-exhibit continuing transmission. While progress towards elimination has been faster in Metema, there remains a problematic hotspot of transmission. Whether migration from Metekel contributes to this is currently unknown. METHODOLOGY/PRINCIPLE FINDINGS: To assess the role of vector migration from Metekel into Metema, we present a population genomics study of 151 adult female vectors using 47,638 RADseq markers and mtDNA CoI sequencing. From additional cytotaxonomy data we identified a new cytoform in Metema, closely related to S. damnosum s.str, here called the Gondar form. RADseq data strongly indicate the existence of two distinctly differentiated clusters within S. damnosum s.l.: one genotypic cluster found only in Metema, and the second found predominantly in Metekel. Because blackflies from both clusters were found in sympatry (in all four collection sites in Metema), but hybrid genotypes were not detected, there may be reproductive barriers preventing interbreeding. The dominant genotype in Metema was not found in Metekel while the dominant genotype in Metekel was found in Metema, indicating that (at the time of sampling) migration is primarily unidirectional, with flies moving from Metekel to Metema. There was strong differentiation between clusters but little genetic differentiation within clusters, suggesting migration and gene flow of flies within the same genetic cluster are sufficient to prevent genetic divergence between sites. CONCLUSIONS/SIGNIFICANCE: Our results confirm that Metekel and Metema represent different transmission foci, but also indicate a northward movement of vectors between foci that may have epidemiological importance, although its significance requires further study.

Interferon regulatory factor 4 plays a pivotal role in the development of aGVHD-associated colitis.

Interferon regulatory factor 4 (IRF4) is a master transcription factor that regulates T helper cell (Th) differentiation. It interacts with the Basic leucine zipper transcription factor, ATF-like (BATF), depletion of which in CD4+ T cells abrogates acute graft-versus-host disease (aGVHD)-induced colitis. Here, we investigated the immune-regulatory role of Irf4 in a mouse model of MHC-mismatched bone marrow transplantation. We found that recipients of allogenic Irf4-/- CD4+ T cells developed less GVHD-related symptoms. Transcriptome analysis of re-isolated donor Irf4-/- CD4+ T helper (Th) cells, revealed gene expression profiles consistent with loss of effector T helper cell signatures and enrichment of a regulatory T cell (Treg) gene expression signature. In line with these findings, we observed a high expression of the transcription factor BTB and CNC homolog 2; (BACH2) in Irf4-/- T cells, which is associated with the formation of Treg cells and suppression of Th subset differentiation. We also found an association between BACH2 expression and Treg differentiation in patients with intestinal GVHD. Finally, our results indicate that IRF4 and BACH2 act as counterparts in Th cell polarization and immune homeostasis during GVHD. In conclusion, targeting the BACH2/IRF4-axis could help to develop novel therapeutic approaches against GVHD.